Ldh kit

After relevant treatment, LDH activity of supernatant was measured by LDH kits (Jiancheng). Cardiomyocytes were detected as follows: cell viability was tested by MTS kit (Promega), caspase-3 activity was detected by caspase-3 activity kit (R&D), apoptosis was measured with Annexin V-EGFP/PI apoptotic detection kit (BD Biosciences, San Diego, CA, USA), and intracellular ROS was assessed using a DCFH-DA probe (Invitrogen, Carlsbad, CA, USA), according to their manufacturer's instructions, respectively [15 (link)].

PVDF (Polyvinylidene Fluoride) hollow fiber materials (aperture 0.2 μm, pure water flux 800 L/m²h under 0.1 Mpa, inner/outer diameter 0.7/1.3 mm) were purchased from SENUO Filtration Technology Co., Ltd. (Tianjin, China). Rat-tail collagen, MTT, Hoechst 33258 dyes were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phalloidine dye was purchased from Yeasen Biotechnology Institute (Shanghai, Chiana). Urea, albumin and LDH kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Fetal Bovine Serum (FBS) and cell lysis buffer were purchased from Beyotime Institute of Biotechnology, Ltd. (Shanghai, China). Sodium nitrite and acrylamide were purchased from Aladdin Reagent (Shanghai, China). All chemical reagents used were of analytical grade.

LDH levels in culture medium and serum were determined using LDH kits (A020-2-2, Nanjing Jiancheng Bioengineering Institute, China) as instructed by the manufacturer.

Briefly, cells were seeded into a 96-well plate at a density of 1 × 10⁵ cells/well in growth medium and cultured to approximately 60–70% confluency, prior to the initiation of experimental treatment. After cell batch and adherence for 4 h, cells were treated with 3 μM Baicalein in 75 mM glucose DMEM. After 36 h of incubation at 37 °C the medium containing the detached cells was collected and centrifuged. The supernatant was used for the assay of LDH activity under the protocol of LDH kits (JianCheng, Nanjing, China). A spectrophotometer was used to measure the optical density (OD) value at 490 nm.

The activities of the antioxidant enzymes SOD and GSH-Px, and the levels of MDA in the heart tissues or H9c2 cell homogenates were determined following the manufacturer’s protocol (Beyotime Biotechnology, Shanghai, China). The activities of lactate dehydrogenase (LDH) both in serum and culture supernate were assessed using the LDH kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the instructions.

Supernatant (0.1 ml) was collected from each group 1 min after reoxygenation to detect LDH activity using LDH kits according to the manufacturer’s instructions (Nanjing Jiancheng Biotech Co. Ltd) as we described (Xu et al., 2013 (link)). LDH activity was expressed as international units per liter (IU/L). N = 6 from three independent experiments.